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Abstract:

The present invention embraces methods for the diagnosis and treatment of
learning or mental disorders, as well as the identification of agents
useful in the treatment of such disorders based upon the identified
involvement of Sarcoplasmic Ca2+-ATPase type 2 Protein in synaptic
plasticity and neurotransmitter release in 22q11 deletion/DiGeorge
Syndrome.

Claims:

1. A method for identifying an agent useful for the treatment of a mental
disorder comprising contacting SERCA2 protein or nucleic acids encoding
the same with a candidate agent and determining whether the candidate
agent inhibits SERCA2 expression or activity thereby identifying an agent
useful for the treatment of a mental disorder.

2. A method for treating a learning disorder or mental disorder
comprising administering to a subject in need of treatment a
therapeutically effective amount of a SERCA inhibitor thereby treating
the subject's learning disorder or mental disorder.

3. The method of claim 2, wherein the SERCA inhibitor is selected from
the group consisting of cyclopiazonic acid, thapsigargin and a
thapsigargin prodrug.

4. The method of claim 2, wherein the mental disorder is a psychiatric
disease.

6. A method for diagnosing susceptibility to developing a learning or
mental disorder comprising obtaining a biological sample from a subject,
and measuring the level of SERCA2 protein or activity in said sample,
wherein an elevated level of SERCA2 protein or activity as compared to a
control indicates an increased susceptibility to developing a learning or
mental disorder.

7. The method of claim 6, wherein the mental disorder is a psychiatric
disease.

9. The method of claim 6, wherein the level of SERCA2 is measured by an
assay selected from the group consisting of protein analysis and
enzymatic analysis.

10. A method for diagnosing a subject suspected of having a learning or
mental disorder comprising measuring the level of synaptic activity of a
subject suspected of having a learning or mental disorder, wherein an
elevated level of neurotransmitter released from presynaptic terminals as
compared to a control subject is indicative of a learning or mental
disorder.

Description:

INTRODUCTION

[0001] This application claims benefit of priority to U.S. Provisional
Application Ser. No. 61/263,872, filed Nov. 24, 2009, the content of
which is incorporated herein by reference in its entirety.

[0004] The orthologous region of the human 22q11.2 locus lies on mouse
chromosome 16. With one exception, all of the human genes in this region
are represented in the mouse, although organized in a different order
(Puech, et al. (1997) Proc. Natl. Acad. Sci. USA 94:14608-14613).
Generation of mouse models that carry chromosomal deficiencies that are
syntenic to the human 22q11.2 microdeletion have been reported (Lindsay,
et al. (1999) Nature 401:379-383; Stark, et al. (2008) Nat. Genet
40:751-760; Merscher, et al. (2001) Cell 104:619-629).

[0006] Although these behavioral and morphological data point to the
hippocampus as a brain region affected during 22q11DS, little is known
about the consequences of 22q11.2 microdeletions on hippocampal synaptic
plasticity, the activity-dependent changes in synaptic efficacy, such as
long-term potentiation (LTP) and long-term depression (LTD) that are
believed to be important for information storage, fine-tuning of synaptic
connections, and learning and memory (Martin, et al. (2000) Annu. Rev.
Neurosci. 23:649-711; Milner, et al. (1998) Neuron 20:445-68). Moreover,
it is not known whether changes in synaptic plasticity and behavior
progress with age in a manner similar to the progression of symptoms in
patients with 22q11.2DS. More importantly, nothing is known about the
molecular mechanisms that are involved in synaptic plasticity and are
affected by 22q11.2 microdeletions.

SUMMARY OF THE INVENTION

[0007] The present invention features a method for identifying an agent
useful for the treatment of a mental disorder by contacting SERCA2
protein or nucleic acids encoding the same with a candidate agent and
determining whether the candidate agent inhibits SERCA2 expression or
activity.

[0008] The present invention also features a method for treating a
learning disorder or mental disorder by administering to a subject in
need of treatment a therapeutically effective amount of a SERCA
inhibitor. In one embodiment, the SERCA inhibitor is selected from the
group consisting of cyclopiazonic acid, thapsigargin and a thapsigargin
prodrug. In other embodiments, the mental disorder is a psychiatric
disease such as schizophrenia, Alzheimer's disease, bipolar disorder,
schizoaffective disorder, DiGeorge syndrome, attention-deficit
hyperactivity disorder, obsessive-compulsive disorder or autism spectrum
disorder.

[0009] An additional feature of this invention is a method for diagnosing
the susceptibility for developing a learning or mental disorder by
obtaining a biological sample from a subject, and measuring the level of
SERCA2 protein or activity in said sample, wherein an elevated level of
SERCA2 protein or activity as compared to a control indicates an
increased susceptibility to developing a learning or mental disorder. In
some embodiments, the mental disorder is a psychiatric disease such as
schizophrenia, Alzheimer's disease, bipolar disorder, schizoaffective
disorder, DiGeorge syndrome, attention-deficit hyperactivity disorder,
obsessive-compulsive or autism spectrum disorder. In other embodiments,
the level of SERCA2 is measured by protein analysis or enzymatic
analysis.

[0010] In addition to the use of SERCA2, the present invention also
features a method for diagnosing a subject suspected of having a learning
or mental disorder by measuring the level of synaptic activity of the
subject, wherein an elevated level of neurotransmitter released from
presynaptic terminals as compared to a control subject is indicative of a
learning or mental disorder.

DETAILED DESCRIPTION OF THE INVENTION

[0011] 22q11 deletion syndrome (22q11DS) is marked early in life by
cognitive deficits, which are compounded during maturation by an
increased risk for development of psychiatric disease, most commonly
schizophrenia. Molecular mechanisms of neuronal dysfunction in 22q11DS
have now been identified using a mouse model of 22q11DS (Df(16)1/+ mice)
that shows mild functional phenotypes early on, but develops a
substantial enhancement in short- and long-term synaptic plasticity at
the hippocampal CA3-CA1 synapse, which coincides with deficits in
hippocampus-dependent spatial memory. These changes are evident in mature
but not young animals. Electrophysiological, two-photon imaging and
glutamate uncaging, and electron microscopic assays in acute brain slices
showed that enhanced neurotransmitter release but not altered
postsynaptic function or structure caused these changes. Enhanced
neurotransmitter release in Df(16)1/+ mice coincided with altered calcium
kinetics in CA3 presynaptic terminals and upregulated sarco(endo)plasmic
reticulum calcium-ATPase type 2 (SERCA2). SERCA inhibitors rescued
synaptic phenotypes of Df(16)1/+ mice. These results indicate that
presynaptic SERCA2 upregulation is a pathogenic event contributing to
cognitive and psychiatric symptoms of 22q11DS such that inhibition of
SERCA2 finds application in alleviating the cognitive symptoms of 22q11DS
and schizophrenia, as well as other learning or mental disorders.
Accordingly, the present invention embraces a method for identifying an
agent useful for the treatment of a mental disorder and methods for
treatment and diagnosis of learning or mental disorders using SERCA2 as a
target.

[0012] For the purposes of the present invention, a SERCA2 protein, also
known as ATPase, Ca(2+)-Transporting, Slow-Twitch (ATP2A2), is intended
to mean a SERCA2 protein from mammals such as mice, rat, bovine, dog or,
most desirably, human. These proteins are known in the art and their
sequences are readily available under GENBANK Accession Nos.
NP--733765 (human), NP--001003214 (dog), XP--612129
(bovine), NP--033852 (mouse), and NP--058986 (rat), which are
incorporated by reference as of the date of filing.

[0013] In accordance with the instant screening assay, a SERCA2 protein or
nucleic acid encoding the same (e.g., in a cell) is contacted with a
candidate agent and it is determined whether the candidate agent inhibits
SERCA2 expression or activity, wherein a candidate agent that inhibits
SERCA2 expression or activity is indicative of an agent useful in the
treatment of a mental disorder. According to the instant screening assay,
changes in SERCA2 expression or activity can be determined biochemically
(e.g., an enzyme kinetic assay), in an assay measuring the expression
level of SERCA2 (protein levels), in an assay measuring physiological
characteristics of a cell (e.g., intracellular Ca2+ levels) and/or
in an assay monitoring changes in behavior of a mammal. As used herein,
"contacting" has its normal meaning and refers to combining two or
more entities (e.g., a protein and a candidate agent, a polynucleotide
and a cell, a cell and a candidate agent, etc.). Contacting can
occur in vitro (e.g., a candidate agent and a cell lysate or isolated
SERCA2 protein are combined in a test tube or other container) or in
vivo (e.g., a candidate agent and an intact test cell are combined in a
container or an animal model is administered the candidate agent).

[0014] An agent that inhibits SERCA2 activity is an agent that binds to
the SERCA2 protein or nucleic acids encoding SERCA2 and reduces, blocks,
or inhibits its expression and/or ability to pump calcium. Agents that
can be assayed in the instant screening method include any substance,
molecule, element, compound, entity, or a combination thereof. It
includes, but is not limited to, e.g., proteins (including antibodies),
oligopeptides, small organic molecules, polysaccharides, polynucleotides
(e.g., DNA or RNA, including polynucleotides encoding a gene product of
interest, or which act as a cell modulator without transcription or
without translation), and the like. It can be a natural product, a
synthetic compound, or a chemical compound, or a combination of two or
more substances. In so far as cyclopiazonic acid, thapsigargin and
thapsigargin prodrugs are known in the art to possess SERCA inhibitory
activity, particular embodiments of this invention encompass screening
analogs of cyclopiazonic acid and thapsigargin for inhibitory activity.
The term "analog" is used herein to refer to a molecule that structurally
resembles a molecule of interest but which has been modified in a
targeted and controlled manner, by replacing a specific substituent of
the reference molecule with an alternate substituent. Compared to the
starting molecule, an analog may exhibit the same, similar, or improved
utility. Synthesis and screening of analogs, to identify variants of
known compounds having improved traits (such as higher potency at a
specific isoform, or higher selectivity at a targeted isoform and lower
activity levels at other isoforms) is an approach that is well-known in
pharmaceutical chemistry. In this respect, particular embodiments of the
present invention embrace an agent that has higher potency and/or higher
selectivity for SERCA2, with limited or no inhibitory activity toward
SERCA1 and/or SERCA3.

[0015] As indicated, SERCA2 activity can be assessed in vitro or in vivo.
By way of illustration, inhibitory activity of a candidate agent can be
assessed by measuring changes in the expression of SERCA2 protein levels.
Detection of SERCA2 protein levels can be achieved with routine methods.
For example, the detection (measurement) of SERCA2 protein levels can be
carried out by using a compound that specifically binds to SERCA2
protein. The detection method (or measurement) is not particularly
limited to this alone. However, the detection (measurement) is desirably
carried out by an immunological technique. In immunological techniques,
an antibody against the SERCA2 is used, and the protein is detected by
using a binding property (binding amount) of the antibody as an
indicator. The term used herein "antibody" includes a polyclonal
antibody, a monoclonal antibody, a chimeric antibody, a single strand
antibody, a CDR graft antibody, a humanized antibody, or the fragment
thereof, and the like. The antibody of the present invention can be
prepared by using an immunological technique, a phage display method, a
ribosome display method, and the like. Examples of the immunological
detection techniques include an ELISA method, radioimmunoassay, FACS, an
immunoprecipitation method, immunoblotting, and the like.

[0016] By way of further illustration, an in vivo assay includes
monitoring changes in intracellular calcium levels. In general, a
calcium-based screen includes contacting the cells of interest with a
candidate agent for a period of time sufficient for the agent to have an
effect in the cell followed by measuring the calcium levels in the cells
and assessing changes in calcium levels in the cell compared to, e.g., a
cell not contacted with the agent. The time sufficient for the agent to
have an effect will depend on the agent(s) being screened, where the
contacting step may take from hours to days (e.g., approximately 1 hour
for small molecule candidate agents, 2 days for siRNA candidate agents).
Measuring the calcium levels can be achieved using any convenient method,
including loading the cells with a calcium dye (e.g., Fura-2) or by
employing cells expressing a fluorescent protein calcium indicator (e.g.,
cameleon). Once calcium levels in the cells are measured and assessed,
agents that intracellular deplete calcium stores are identified as
inhibitors of SERCA2 activity. Cells used in such screening assays can be
any mammalian cell, including primary cells, transformed cells,
genetically modified cells, etc. In certain embodiments, the cells are
human cells (e.g., HeLa cells). The origin of cells is not particularly
limited. However, it is preferable to use cells derived from the central
nervous system tissue. Among the central nervous system tissue, it is
preferable to use cells derived from the prefrontal cortex of the
forebrain, the nucleus accumbens, the striatum, the midbrain, or the
hippocampus.

[0017] Agents identified in the instant screening assay as inhibiting
SERCA2 activity find use in the study of SERCA2 activity as well as in
the prevention or treatment of diseases or conditions in which SERCA2
activity is implicated. In this respect, agents identified in the instant
screening assay can be formulated in pharmaceutical compositions suitable
for administration to a subject in need of treatment. Such compositions
typically contain from about 0.1 to 90% by weight (such as 1 to 20% or 1
to 10%) of the SERCA2 inhibitor in a pharmaceutically acceptable carrier.
A pharmaceutically acceptable carrier is a material useful for the
purpose of administering the medicament, which is preferably sterile and
non-toxic, and can be solid, liquid, or gaseous materials, which is
otherwise inert and medically acceptable, and is compatible with the
active ingredients. A generally recognized compendium of methods and
ingredients of pharmaceutical compositions is Remington: The Science and
Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott
Williams & Wilkins: Philadelphia, Pa., 2000.

[0019] Liquid compositions for oral administration prepared in water or
other aqueous vehicles can include solutions, emulsions, syrups, and
elixirs containing, together with the active compound(s), wetting agents,
sweeteners, coloring agents, and flavoring agents. Various liquid and
powder compositions can be prepared by conventional methods for
inhalation into the lungs of the subject to be treated.

[0020] Injectable compositions may contain various carriers such as
vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate,
ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol,
propylene glycol, liquid polyethylene glycol, and the like). For
intravenous injections, the compounds may be administered by the drip
method, whereby a pharmaceutical composition containing the active
agent(s) and a physiologically acceptable excipient is infused.
Physiologically acceptable excipients may include, for example, 5%
dextrose, 0.9% saline, Ringer's solution or other suitable excipients.
For intramuscular preparations, a sterile composition of a suitable
soluble salt form of the compound can be dissolved and administered in a
pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5%
glucose solution, or depot forms of the compounds (e.g., decanoate,
palmitate, undecylenic, enanthate) can be dissolved in sesame oil.
Alternatively, the pharmaceutical composition can be formulated as a
chewing gum, lollipop, or the like.

[0021] As indicated, SERCA inhibitors find application in the treatment or
learning or mental disorders. In this respect, the present invention also
embraces a method for treating a learning disorder or mental disorder by
administering to a subject with a learning disorder or mental disorder a
therapeutically effective amount of a SERCA inhibitor. As used herein,
"treating" or "treatment" of a disease refers to arresting, ameliorating
or delaying the onset of a disease, disorder, or at least one clinical
symptom or physical parameter of a disease or disorder, which may or may
not be discernible by the patient. In certain embodiments, "treating" or
"treatment" refers to inhibiting or controlling the disease or disorder,
either physically (e.g., stabilization of a discernible symptom),
physiologically (e.g., stabilization of a physical parameter), or both.
Effectiveness of the a SERCA inhibitor can be determined by measuring or
monitoring SERCA expression or activity.

[0022] Subjects benefiting from treatment in accordance with the instant
method include those having, those suspected of having or those
predisposed to have (e.g., genetic predisposition) a learning disorder or
mental disorder. Learning disorders include childhood learning disorders,
wherein the subject has an impaired ability to learn. Such learning
disorders can be diagnosed by using the DSM-IV criteria (APA, 1994,
Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition),
Washington, D.C.). Mental disorders embraced by the present invention
include, but are not limited to psychiatric diseases such as
schizophrenia, Alzheimer's disease, bipolar disorder, schizoaffective
disorder, DiGeorge syndrome, attention-deficit hyperactivity disorder,
obsessive-compulsive disorder and autism spectrum disorder.

[0023] As used herein, the term "schizophrenia" refers to a psychiatric
disorder that includes at least two of the following: delusions,
hallucinations, disorganized speech, grossly disorganized or catatonic
behavior, or negative symptoms. Subjects can be diagnosed as
schizophrenic using the DSM-IV criteria.

[0024] The term "Alzheimer's Disease" refers to a progressive mental
deterioration manifested by memory loss, confusion and disorientation
beginning in late middle life and typically resulting in death in five to
ten years. Pathologically, Alzheimer's Disease can be characterized by
thickening, conglutination, and distortion of the intracellular
neurofibrils, neurofibrillary tangles and senile plaques composed of
granular or filamentous argentophilic masses with an amyloid core.
Methods for diagnosing Alzheimer's Disease are known in the art. For
example, the National Institute of Neurological and Communicative
Disorders and Stroke-Alzheimer's Disease and the Alzheimer's Disease and
Related Disorders Association (NINCDS-ADRDA) criteria can be used to
diagnose Alzheimer's Disease (McKhann, et al. (1984) Neurology
34:939-944). The patient's cognitive function can be assessed by the
Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog; Rosen,
et al. (1984) Am. J. Psychiatry 141:1356-1364).

[0025] Bipolar disorder, also known as manic depressive disorder, manic
depression or bipolar affective disorder, is a psychiatric diagnosis that
describes a category of mood disorders defined by the presence of one or
more episodes of abnormally elevated mood clinically referred to as mania
or, if milder, hypomania. Individuals who experience manic episodes also
commonly experience depressive episodes or symptoms, or mixed episodes in
which features of both mania and depression are present at the same time.
These episodes are usually separated by periods of "normal" mood, but in
some individuals, depression and mania may rapidly alternate, known as
rapid cycling. Extreme manic episodes can sometimes lead to psychotic
symptoms such as delusions and hallucinations. Subjects can be diagnosed
as having bipolar disorder using the DSM-IV-TR criteria and the World
Health Organization's International Statistical Classification of
Diseases and Related Health Problems, the ICD-10.

[0026] Schizoaffective disorder is a psychiatric diagnosis that describes
a mental disorder characterized by recurring episodes of mood disorder
and psychosis. Distortions in perception alternate with and occur
simultaneously with elevated or depressed mood. These perceptual
distortions may affect all five senses, including sight, hearing, taste,
smell and touch, but most commonly manifest as auditory hallucinations,
paranoid or bizarre delusions, or disorganized speech and thinking with
significant social or occupational dysfunction. Subjects can be diagnosed
as having a schizoaffective disorder using the DSM-IV-TR criteria.

[0027] Characteristic signs and symptoms of DiGeorge syndrome may include
birth defects such as congenital heart disease, defects in the palate,
most commonly related to neuromuscular problems with closure
(velo-pharyngeal insufficiency), learning disabilities, mild differences
in facial features, and recurrent infections. DiGeorge syndrome may be
first spotted when an affected newborn has heart defects or convulsions
from hypocalcemia due to malfunctioning the parathyroid glands and low
levels of parathyroid hormone (parathormone). Affected individuals may
also have any other kind of birth defect including kidney abnormalities
and significant feeding difficulties as babies. Autoimmune disorders such
as hypothyroidism and hypoparathyroidism or thrombocytopenia (low
platelet levels), and psychiatric illnesses are common late-occurring
features.

[0028] The term "attention-deficit hyperactivity disorder attention
deficit disorder," as used herein, refers to a disorder that is most
commonly exhibited by children and which can be characterized by
increased motor activity and a decreased attention span. The DSM-IV
criteria can be used to diagnose attention deficit disorder.

[0029] Obsessive-compulsive disorder (OCD) is a mental disorder
characterized by intrusive thoughts that produce anxiety, by repetitive
behaviors aimed at reducing anxiety, or by combinations of such thoughts
(obsessions) and behaviors (compulsions). The symptoms of this anxiety
disorder range from repetitive hand-washing and extensive hoarding to
preoccupation with sexual, religious, or aggressive impulses as well as
corrections of minor things. These symptoms can be alienating and
time-consuming, and often cause severe emotional and economic loss.
Although the acts of those who have OCD may appear paranoid and come
across to others as psychotic, OCD sufferers often recognize their
thoughts and subsequent actions as irrational, and they may become
further distressed by this realization.

[0030] As used herein, the term "autism spectrum disorder" refers to a
spectrum of psychological conditions characterized by widespread
abnormalities of social interactions and communication, as well as
severely restricted interests and highly repetitive behavior. Subjects
with autism experience mental introversion characterized by morbid
self-absorption, social failure, language delay, and stereotyped
behavior. The three main forms of ASD are Autism, Asperger syndrome,
Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS),
sometimes called atypical autism. Patients can be diagnosed as suffering
from autism by using the DSM-IV criteria.

[0031] Subjects to be treated in accordance with the instant method can be
provided with a therapeutically effective amount of a SERCA inhibitor
identified by the instant screening assay or agents known in the art to
exhibit SERCA inhibitory activity. For example, cyclopiazonic acid,
thapsigargin and thapsigargin prodrugs have been shown to inhibit SERCA
activity. In addition, phospholamban is a known, endogenous,
muscle-specific SERCA2 inhibitor (Luo, et al. (1994) Circ. Res.
75(3):401-409. Accordingly, analogs and prodrugs of these agents are
embraced by the instant method. In some embodiments, the agent selected
for treatment has a higher potency and/or higher selectivity for SERCA2,
with limited or no inhibitory activity toward SERCA1 and/or SERCA3.

[0032] For the purposes of the present invention, "therapeutically
effective amount" refers to the amount of a compound that, when
administered to a subject for treating a disease, or disorder, or at
least one of the clinical symptoms of a disease or disorder, is
sufficient to affect such treatment of the disease, disorder, or symptom.
The "therapeutically effective amount" may vary depending, for example,
on the compound, the disease, disorder, and/or symptoms of the disease,
severity of the disease, disorder, and/or symptoms of the disease, the
age, weight, and/or health of the patient to be treated, and the judgment
of the prescribing physician. An appropriate amount in any given instance
may be readily ascertained by those skilled in the art or capable of
determination by routine experimentation. Generally, treatment can be
provided for at least several weeks to several years or life-long as
needed. In accordance with the methods of the invention, appropriate
dosages of SERCA inhibitor can readily be determined by those of ordinary
skill in the art of medicine by monitoring the patient for signs of
disease amelioration or inhibition, and increasing or decreasing the
dosage and/or frequency of treatment as desired.

[0033] Where appropriate, a pharmaceutical composition containing a SERCA
inhibitor can be administered to a subject suffering from learning or
mental disorder along with, or in sequence with, an art-known drug for
treating the learning or mental disorder. For example, art-known drugs
for treating schizophrenia, include olanzapine, clozapine, haloperidol,
and the like. Similarly, a SERCAtrams inhibitor can be used in
combination with, or in sequence with, other art-known antipsychotics
(e.g., "typical," "atypical," and depot antipsychotics for treating
schizophrenia and other psychotic conditions), psychostimulants (for
treating attention deficit disorder or learning disorders), or
Alzheimer's disease therapeutics (for treating Alzheimer's disease). Such
pharmaceutical compositions are included within the invention. In
general, the antipsychotic, psychostimulant, or Alzheimer's disease
therapeutic typically is administered at a dosage of 0.25-5000 mg/d
(e.g., 5-1000 mg/d)). "Typical" antipsychotics are conventional
antipsychotics such as phenothiazine, butryophenones, thioxantheses,
dibenzoxazepines, dihydroindolones, and diphenylbutylpiperidines.
"Atypical" antipsychotics are a new generation of antipsychotics which
generally act on the dopamine D2 and 5HT2 serotonin receptor
and have high levels of efficacy and a benign extrapyramidal symptom side
effect profile. Examples of typical antipsychotics include
Chlorpromazine, Thioridazine, Mesoridazine, Fluphenazine, Perphenazine,
Trifluoperazine, Thiothixene, Haloperidol, Loxapine, Molindone,
Acetophenazine, Droperidol, Pimozide. Examples of atypical antipsychotics
include Clozapine, Risperidone, Olanzapine, and Quetiapine. Depot
antipsychotics also can be used, e.g., Haloperidol decanoate,
Fluphenazine decanoate, and Fluphenazine enanthate. Additional
antipsychotics include Butaperazine, Carphenazine, Remoxipride,
Piperacetazine, Sulpiride, and Ziprasidone. Psychostimulants that are
particularly useful for treating attention deficit disorder include
Dextroamphetamine, Methamphetamine, Methylphenidate, and Pemoline.
Examples of Alzheimer's disease therapeutics that can be used in the
invention include Donepezil and Tacrine. Thus, the invention also
provides pharmaceutical compositions that contain one or more SERCA2
inhibitors along with an antipsychotic, psychostimulant, or Alzheimer's
disease therapeutic.

[0034] In addition to, or as an alternative to, conventional methods of
diagnosing a subject for a learning disorder or mental disorder, the
present invention also embraces a method for diagnosing an increased
susceptibility to developing a learning or mental disorder based upon
SERCA2 levels. In accordance with the diagnostic method of the invention,
a biological sample is obtained from a subject to be tested, and the
level of SERCA2 in said sample is measured and compared to a control,
wherein an elevated level of SERCA2 as compared to the control indicates
an increased susceptibility to developing a learning or mental disorder.

[0035] Insofar as SERCA2 is ubiquitous, biological samples in which the
level of SERCA2 is to be measured include biopsy samples (e.g., brain
tissue) or bodily fluids (e.g., blood). The level of SERCA2 can be
determined at the protein level (including amount or activity of SERCA2),
wherein the level of SERCA2 as compared to a control is indicative of the
level, extent, or severity of disease. For example, a slight increase in
SERCA2 as compared to a control is indicative of a mild disease. In
particular embodiments, the instant diagnostic assay is carried out my
measuring the level or activity of SERCA2 protein. Controls can include
relative or absolute amounts of SERCA2 protein or activity levels in a
subject or population of subjects without a learning or mental disorder.

[0036] In addition to protein analysis and enzymatic analysis, some
embodiments embrace genetic analysis, wherein mutations including
polymorphisms and DNA modifications (e.g., methylation marks and the
like), in the SERCA2 gene are associated with an elevated level of SERCA2
protein or activity and a learning or mental disorder. Such mutations are
identified by comparing the nucleotide sequence of the gene encoding
SERCA2 in subjects with a learning or mental disorder to the nucleotide
sequence of subjects without a learning or mental disorder and
establishing a correlation between one or more mutations with an elevated
level of SERCA2 protein or activity and the learning or mental disorder.

[0037] In addition, the present invention embraces a method for diagnosing
a subject suspected of having a learning or mental disorder by measuring
the level of synaptic activity of a subject suspected of having a
learning or mental disorder, wherein an elevated level of
neurotransmitter released from presynaptic terminals as compared to a
control subject is indicative of a learning or mental disorder. Levels of
synaptic activity can be determined according to the methods exemplified
herein or any other method conventionally employed in the art. Subjects
serving as controls include those with normal or wild-type levels
synaptic activity or a subject with a predetermined amount of synaptic
activity.

[0038] The diagnostic methods of the invention can be used in the initial
determination of whether a subject has a learning or mental disorder or
in the confirmation of a diagnosis based upon conventional behavioral or
clinical analysis. In this respect, subjects benefiting from the instant
diagnostic methods include those suspected of having, or those
predisposed (e.g., based upon heredity) to have a learning or mental
disorder.

[0039] As with the method of treatment, learning or mental disorders that
can be diagnosed in accordance with the instant diagnostic methods
include, but are not limited to, having, suspected of having or those
predisposed to have schizophrenia, Alzheimer's disease, bipolar disorder,
schizoaffective disorder, DiGeorge syndrome, attention-deficit
hyperactivity disorder, obsessive-compulsive disorder or autism spectrum
disorder.

[0040] The invention is described in greater detail by the following
non-limiting examples.

Example 1

Materials and Methods

[0041] Animals.

[0042] Young (6-8 weeks) and mature (16-20 weeks) Df(16)1/+ male and
female mice and their respective gender-controlled wild-type (WT)
littermates were used. Mice were maintained on the C57BL/6 genetic
background for at least 9 generations.

[0046] The field recordings were performed using a setup with eight
submerged recording chambers (Campden Instruments, Lafayette, Ind.).
Recordings in each chamber were performed independently. Field excitatory
postsynaptic potentials (fEPSPs) from the CA1 stratum radiatum were
recorded using an extracellular glass pipette (3-5 MΩ) filled with
ACSF. Schaffer collateral fibers in the s. radiatum were stimulated with
a bipolar tungsten electrode placed 200 to 300 μm away from the
recording pipette. Stimulation intensities were chosen to produce a fEPSP
with a 0.5 V/s slope. Paired-pulse facilitation (PPF) experiments were
performed using a pair of stimuli of the same intensity delivered 20, 50,
100, 200, and 1000 ms apart.

[0047] LTP was induced by three periods of 200-Hz tetanization delivered
every 5 minutes. Every period of tetanization was composed of 10 trains
of 200-Hz stimulation delivered at the same intensity for 200 ms (40
stimulations) every 5 seconds. A similar protocol has previously been
used to induce compound (presynaptic and postsynaptic) LTP at CA3-CA1
synapses in the hippocampus (Zakharenko, et al. (2001) Nat. Neurosci.
4:711-7; Cavus & Teyler (1996) J. Neurophysiol. 76:3038-47; Zakharenko,
et al. (2003) Neuron 39:975-90).

[0048] Whole-Cell Electrophysiology.

[0049] Whole-cell recordings were obtained from the cell bodies of CA1 and
CA3 neurons. For current-clamp recordings, patch pipettes (open pipette
resistance, 3-5 MO) were filled with an internal solution containing 140
mM KMeSO4, 8 mM NaCl, 1 mM MgCl2, 10 mM HEPES, 5 mM MgATP, 0.4
mM Na2 GTP, 300 μM Fluo 5F, and 10 to 25 μM ALEXA 594 (pH
7.3). For voltage-clamp recordings, the potassium-based solution was
replaced with a cesium-based internal solution. Whole-cell recordings
were registered using a Multiclamp 700B (Molecular Devices, Sunnyvale,
Calif.), digitized (10 kHz; DigiData 1322A, Molecular Devices), and
recorded using pCLAMP 9.0 software (Molecular Devices). Spontaneous
miniature excitatory postsynaptic currents (mEPSCs) were recorded at -70
mV holding potential in the presence of picrotoxin (100 μM) and
tetrodotoxin (1 μM) in the extracellular solution for at least 1 hour.
Amplitude, 10% to 90% rise time, decay time constant, and interevent
intervals of mEPSCs were analyzed off-line using the Mini-Analysis
Program (Synaptosoft Inc., Leonia, N.J.). All detected events were
verified visually, and events with amplitudes less than 5 pA were
rejected. Evoked excitatory postsynaptic currents (EPSCs) were recorded
in the presence of QX-314 (5 mM) in the intracellular solution to block
the generation of back-propagating action potentials (APs) and picrotoxin
in the extracellular solution to block inhibitory transmission. EPSCs
were evoked at 0.1 Hz with a bipolar electrode placed in the s. radiatum
200 to 300 μm from the recording pipette and 100 to 150 μm from the
soma. The amplitude of stimulation was adjusted to evoke 100 pA EPSCs at
-70 mV. To determine the average amplitude, rise and decay times, 10 to
20 EPSCs (interstimulus interval >10 seconds) were collected from each
neuron. In whole-cell short-term plasticity (STP) experiments, neurons
were held at -70 mV, and 10 stimulations at different frequencies were
applied to Schaffer collaterals. Data were analyzed by normalizing all
EPSCs in the train to the amplitude of the first EPSC. The current ratio
of AMPA receptors to NMDA receptors (AMPAR/NMDAR) was calculated from the
EPSC traces recorded at +40 mV. The amplitude of stimulation was adjusted
to evoke 50 pA EPSCs at -70 mV. The AMPAR current was determined at time
points when EPSCs recorded at -70 mV reached their peaks, and the NMDAR
currents were determined 100 ms after the peaks. EPSCs were analyzed
off-line using Clampfit 10.1 software (Molecular Devices).

[0052] ALEXA 594 fluorescence (R, red channel) was used to image and
reconstruct dendritic morphology of CA1 neurons and axonal morphology of
CA3 neurons. ImageJ was used to analyze dendritic branching and
morphology of dendritic spines. Changes in Fluo 5F fluorescence (G, green
channel) were used to visualize changes in Ca2+ concentrations in
dendritic spines and presynaptic terminals. Synaptically evoked changes
in the fluorescence of Fluo 5F were measured in current-clamp using
line-scan mode (500 Hz) in dendritic spines. Fluorescence changes were
quantified as an increase in Fluo 5F fluorescence normalized to the
average ALEXA 594 fluorescence (ΔG/R) (Yasuda R, et al. (2004) Sci.
STKE 2004:15). To identify synaptic inputs, multiple scans were taken
through the apical part of secondary or tertiary dendrites (50-150 μm
from a soma) of CA1 neurons in current-clamp mode in response to synaptic
stimulation. Stimulation intensity was adjusted to evoke 50 to 100 pA in
voltage-clamp mode. Dendritic sites responding with maximal Fluo 5F
transients to synaptic stimulation were chosen for imaging, and line
scans through corresponding dendritic spines were taken. Calcium
transients were measured in a single dendritic site on each recorded
neuron. Ca2+ transients in CA3 presynaptic terminals were recorded in a
similar fashion. Axons were identified based on ALEXA 594 fluorescence as
thin processes emanating from cell bodies, which have no dendritic
spines. Presynaptic terminals were identified as boutons situated along
axons. Ca2+ transients in presynaptic boutons were recorded in line-scan
mode and were evoked by injection of a depolarizing current (0.5 ms,
1.2-2.5 nA) that evoked an AP in the recorded neuron. The number of
evoked APs was controlled online in current-clamp mode. Between 2 to 4
boutons were recorded on each neuron and the calcium transients in each
bouton were measured independently. When 40 APs at 200 Hz were delivered
to a presynaptic bouton, the following precautions were taken: it was
monitored that 40 pulses evoked 40 APs in a recorded neuron, and Fluo 5F
fluorescence in presynaptic terminals did not saturate. Following
delivery of 40 APs at 200 Hz, neurons were depolarized to +20 mV and the
Fluo 5F fluorescence in the same bouton was measured. The maximal
increase in Fluo 5F fluorescence in presynaptic boutons was 63%±18%
higher during +20 mV depolarization than that during 200-Hz tetanization
(p=0.03, n=3), indicating that the dye was not saturated during these
experiments.

[0053] FM 1-43 Assay in Acute Hippocampal Slices.

[0054] The FM 1-assay was performed as a modification of a method
established in the art (Zakharenko, et al. (2003) Neuron 39:975-90;
Zakharenko, et al. (2002) Neuron 35:1099-1110). FM 1-43 (10 μM) was
washed into acute slices for 20 to 30 minutes. Loading of presynaptic
boutons was performed by using 10-Hz synaptic stimulation for 2 minutes
in the presence of D-APV (50 μM; Tocris Bioscience, Ellisville, Mo.)
to avoid synaptic plasticity. ADVASEP-7 (200 μM; Biotium Hayward,
Calif.) was then washed in for 20 to 30 minutes to remove the
extracellular FM 1-43 dye. Loaded presynaptic boutons were visualized
using TPLSM (900 nm). A series of four images at different focal planes
was acquired every 5 seconds. Each image was 512×512 pixels
(33.6×33.6 μm), 0.066 μm/pixel in the x-y axes, and images
were separated by 1-μm steps in the z direction.

[0055] Images in each z-section series were aligned and analyzed using
custom software written in Interactive Data Language (IDL, ITT Visual
Information Solutions, Boulder, Colo.). The area of the slice in which
fluorescent puncta were analyzed was restricted to the area within 50 to
150 μm from the pyramidal layer and corresponded to the areas of the
apical dendrites where Ca2+ experiments were conducted. Images showing
projections of maximal z-axis intensity were made for each subset of a
given stack. Puncta were initially identified by a semi-automated
procedure written in IDL based on 2 criteria: (1) fluorescence intensity
greater than 2× standard deviations above the mean background and
(2) diameter between 0.3 to 1.8 μm.

[0056] Fluorescence measurements were made by spatially averaging signals
over a region centered over each of the identified puncta for each time
point during the unloading protocol. Images at successive time points
were checked for overlap to help track puncta, which underwent small
random movements. Puncta that underwent considerable lateral movement or
more than 20% loss of their fluorescent intensity without synaptic
stimulation due to photobleaching were excluded from the analysis. The
spatially averaged, activity-dependent fluorescence intensity of each
punctum obtained at each time point during the unloading procedure was
then normalized by the initial fluorescence intensity of that punctum
following the FM 1-43-loading procedure prior to unloading. The unloading
procedure included 10 trains of tetanic synaptic stimulations (same as
the LTP-induction protocol). Each train included 40 stimulations
delivered at 200 Hz. This unloading procedure followed by 10-Hz
stimulation was applied for 2 minutes to maximally release FM 1-43 from
boutons. Decay of intensity during the 200-Hz unloading procedure was
fitted to a single exponential function by using a custom-made routine
written in IDL, and FM 1-43 destaining half-time (t1/2) for every punctum
was calculated. The rate of destaining for each punctum was expressed as
1/t1/2. Puncta for which fluorescence intensities during unloading could
not be fitted to a single exponential function were not included in the
analysis.

[0057] Two-Photon Glutamate Uncaging.

[0058] MNI-glutamate (2.5 mM; Tocris Bioscience) was added to the
recording ACSF. MNI-glutamate was uncaged by using TriggerSync (Prairie
Technologies) and by 0.2- to 0.5-ms pulses that were delivered from a
second Ti:sapphire Chameleon Ultra femtosecond-pulsed laser (Coherent
Inc.) at 720 nm. The intensity and duration of the uncaging laser was
adjusted to mimic mEPSPs (0.4-0.5 mV) or mEPSCs (10-12 pA). In all
experiments, before each uncaging pulse, an image of the spine was
acquired and automatically aligned with a reference image of the spine.
The uncaging laser intensity was normalized to the same degree of ALEXA
594 bleaching by using the previously described method to deliver the
same photostimulation power to individual dendritic spines, independent
of the depth of the spine in the slice or the refraction index of local
tissue (Bloodgood & Sabatini (2005) Science 310:866-869). Once the
duration and laser power were adjusted, 6 to 9 test pulses were delivered
around the perimeter of a spine head to determine the optimal site of
uncaging (determined as the maximal response). Another test pulse to the
center of the spine head determined the level of ALEXA 594 bleaching.
MNI-glutamate was then uncaged at the optimal site. The level of ALEXA
594 bleaching was used to adjust the laser power (but not duration) for
other dendritic spines of the same neuron. The point-spread function of
the focal volume of two-photon excitation was 300 nm laterally and 1100
nm axially (NA 0.9) based on images of 100-nm fluorescent beads.

[0059] Spatial Memory Testing.

[0060] Spatial memory was tested in the Morris water maze. A circular
steel water maze (diameter, 4 feet; depth, 2 feet) filled with water
(room temperature) clouded with white, nontoxic, water-based paint was
used. Compass points labeled along the rim served as trial starting
positions. For the spatial tasks, water levels were raised 0.25 in above
the clear, PLEXIGLASS escape platform. For the nonspatial task, the water
level was lowered so that the escape platform was visible 0.25 inch above
the water's surface. The water maze environment was full of visual cues
whose locations remained fixed throughout the learning protocol. Mouse
movements in the maze were recorded using a video camera tracking system
(HVS Image, Co., Buckingham, UK) mounted above the pool, and path length
was measured. Animals learned to find a hidden platform in the training
(TRA) quadrant using the standard spatial version of the Morris water
maze task for successive days. Each day, animals were given four 1-minute
trials from each starting position with an intertrial latency of at least
60 seconds. The order of starting locations was counterbalanced each day
using a Latin square design.

[0061] A spatial memory (probe) trial was administered on the day
following the completion of spatial learning. With the platform removed,
animals received a single 1-minute trial in which the animal tried to
find the escape platform in the TRA quadrant. This trial started from the
point that was the farthest from the platform's location on the previous
training day. The overall path length was measured for each mouse, and
the relative path length for each quadrant was calculated.

[0062] Mice started nonspatial learning tasks at least 8 days after
completion of the spatial protocol. In this task, the platform was
visible above the water's surface. Animals were trained using the
standard nonspatial version of the Morris water maze task for 5
successive days. During training day 1, they saw the escape platform
located in the same position used during spatial training. Each day
thereafter, the escape platform was rotated, in a clockwise manner, to
the next quadrant. Each day, animals were given four 1-minute trials in
the same manner that occurred during spatial training.

[0063] Electron Microscopy.

[0064] Hippocampal slices were fixed in 2.5% glutaraldehyde in 0.1 M
sodium cacodylate buffer, thrice rinsed in the same buffer, and
dehydrated in a graded series of alcohol and then propylene oxide washes.
The tissue was infiltrated and embedded in Epon-Araldite and polymerized
overnight at 70° C. Seventy-nm sections were cut on a LEICA UC6
ultramicrotome fitted with a Diatome diamond knife and stained with lead
citrate and 8% uranyl acetate. Thick sections were trimmed to a region
between the CA1 and dentate gyrus cell body layers, and the somata were
used as guideposts to find the CA1 area where synapses were identified.
The sections were imaged on a JEOL 1200EX11 transmission electron
microscope with an AMT XR111 megapixel digital camera. Synapses were
counted as regions of membrane enclosing synaptic vesicles in close
proximity to a postsynaptic density. Vesicle size and number and
postsynaptic density length were measured in ImageJ by tracing with the
line or elliptical selection tools followed by measurement.

Dysregulation of Presynaptic Calcium and Synaptic Plasticity in a Mouse
Model of 22q11 Deletion Syndrome

[0073] Hippocampal LTP is Enhanced in Mature but not in Young Df16(1)/+
Mice.

[0074] To examine whether the Df(16)1 deletion affects LTP at excitatory
CA3-CA1 synapses, fEPSPs were recorded before and after the delivery of a
200-Hz tetanus to the Schaffer collaterals in acute brain slices from WT
and mutant mice. Because this induction protocol potentiates both
neurotransmitter release and postsynaptic responses at CA3-CA1 synapses
(Bayazitov, et al. (2007) supra; Zakharenko, et al. (2001) Nat. Neurosci.
4:711-7), it was reasoned that it would reveal changes in both
presynaptic and postsynaptic components of LTP. Because patients with
22q11DS manifest a decline in cognitive function (Gothelf, et al. (2007)
Schizophr. Res. 96:72-81), LTP was teste in mice of two different ages.
It was found that LTP was not substantially altered in younger (6-8
weeks) Df(16)1/+ mice compared to WT littermates (p=0.174, 43-45 slices,
8 mice per genotype). In contrast, more mature (16-20 weeks) Df(16)1/+
mice exhibited dramatically enhanced post-tetanic potentiation (PTP) and
LTP. The PTP of fEPSPs measured 5 minutes after tetanization (fEPSP5) was
approximately 120% higher in mature Df(16)1/+ mice than in WT mice,
increasing to 347.9%±35.3% over baseline, as compared to 156%±14.6%
in WT littermates (p=0.000002, 24-slices, 6-8 mice). In mature WT mice,
LTP of fEPSPs measured 6 hours post-tetanus (fEPSP360) showed a
39.3%±10.5% increase over baseline; whereas in Df(16)1/+ littermates,
the fEPSP360 was approximately 200% higher than in WT mice and showed a
118.4%±19.7% increase over baseline (p=0.0005, 24-29 slices, 6-8
mice). Changes in LTP were not due to an increase in the number of
stimulated afferents, because no changes in fiber volley were detected in
mature Df(16)1/+ or WT mice.

[0075] Along with developmental changes in LTP, Df(16)1/+ mice showed
age-dependent deficits in the hippocampus-dependent spatial behavioral
task, the Morris water maze. Young Df(16)1/+ mice did not show any
difference in spatial memory as compared to their WT littermates.
However, mature Df(16)1/+ mice showed deficient spatial memory, whereas
spatial learning and nonspatial memory remained intact. These results
demonstrate that Df(16)1/+ mice develop a deficit in
hippocampus-dependent spatial memory that coincides with the onset of LTP
abnormalities.

[0076] To determine the cause of the substantial increase in LTP in mature
Df(16)1/+ mice, differences in basal synaptic transmission were analyzed.
However, input-output coupling at CA3-CA1 synapses did not significantly
differ between mature Df(16)1/+ mice and WT littermates (p>0.05, 24-29
slices, 6-8 mice). Similarly, single-cell recording revealed no
differences in spontaneous or evoked EPSCs in mature Df(16)1/+ mice.
Amplitudes of spontaneous miniature EPSCs (mEPSCs) (18.01±1.28 pA for
Df(16)1/+ and 17.73±1.03 pA for WT, p=0.87, 6-7 neurons, 551-2445
events per neuron), as well as intervals between mEPSCs (4.78±0.77
seconds for Df1(16)/+ and 3.81±0.74 seconds for WT, p=0.135, 6-7
neurons) were not significantly different between the genotypes. Rise
times (2.39±0.11 ms for Df1(16)/+ and 2.47±0.14 ms for WT mice,
p=0.29) and decay times (6.38±0.22 ms for Df(16)1/+ and 6.50±0.34
ms for WT mice, p=0.25, 6-7 neurons) of mEPSCs were also not different in
mutant and WT mature mice. Similarly, rise times and decay times of EPSCs
evoked by a single synaptic stimulation were not significantly different
between mature Df(16)1/+ and WT littermates (p=0.29 and 0.49,
respectively, 10 neurons per genotype). These data indicate that basal
synaptic transmission is normal at excitatory synapses of Df(16)1/+ mice,
whereas LTP mechanisms undergo substantial developmental changes in the
Df(16)1/+ model of 22q11DS.

[0078] Previous studies have reported structural abnormalities in the
brains of 22q11DS mouse models (Meechan, et al. (2009) Proc. Natl. Acad.
Sci. USA 106:16434-16445; Stark, et al. (2008) Nat. Genet. 40:751-760;
Mukai, et al. (2008) Nat. Neurosci. 11:1302-1310); thus, it was
determined whether developmental changes in synapse structure contribute
to the enhanced LTP in mature Df(16)1/+ mice. Dendritic structures were
visualized by loading CA1 pyramidal neurons of mature mice with the
fluorescent dye ALEXA 594 through a whole-cell pipette and imaging them
using TPLSM. The overall morphology of apical dendritic trees of CA1
neurons from mature Df(16)1/+ mice was indistinguishable from that of WT
littermates, and quantification of branching by Scholl analysis revealed
no difference between genotypes (p=0.863, 7-9 neurons). Furthermore, the
length (p=0.21), width (p=0.84) and density (p=0.36) of dendritic spines
were similar between mutants and WT littermates (5-7 neurons, 27-28
dendrites, 1171-1290 spines). Electron microscopy was also used to
resolve synapses and subsynaptic structures in this region. However, this
analysis revealed no abnormalities in the number of CA1 synapses, the
number of vesicles, or the size of the postsynaptic densities of mature
Df(16)1/+ mice. The only significant change was a slight increase in
synaptic vesicle diameter in Df(16)1/+ synapses (30.97±0.27 nm)
compared to that of WT synapses (29.18±0.31 nm, 60-80 synapses,
p=0.005). However, this change did not affect neurotransmitter release,
because the amplitude of spontaneous mEPSCs was not affected in mature
Df(16)1/+ mice.

[0079] Similarly, electrophysiological characteristics of postsynaptic
neurons were not altered in mature Df(16)1/+ mutants. AMPAR/NMDAR ratios
measured in CA1 neurons of Df(16)1/+ and WT mice were not significantly
different (p=0.53, 9-11 neurons). There was no difference in the resting
membrane potentials (-65.8±0.8 mV for WT and -66.2±1.1 mV for
Df(16)1/+ mice, 15 neurons per genotype, p=0.78) or in the excitability
of CA1 neurons in mature WT and Df(16)1/+ mice. Injection of depolarizing
currents evoked a similar number of APs (p=0.75) at similar threshold
membrane potentials (p=0.67, 18-19 neurons) in mutant and WT mice. Thus,
these data indicate that morphological or functional changes in
postsynaptic CA1 neurons do not contribute to the enhancement of LTP at
CA3-CA1 synapses in mature Df1(16)/+ mice.

[0080] Short-Term Synaptic Plasticity is Enhanced in Mature but not Young
Df16(1)/+ Mice.

[0081] To investigate the presynaptic contribution to the Df(16)1/+
phenotype, short-term synaptic plasticity, which relies primarily on the
presynaptic machinery, was tested. To measure PPF of fEPSPs, field
potentials evoked by 2 synaptic stimulations delivered at different
interpulse intervals were recorded. It was found that PPF in slices from
mature Df(16)1/+ mice was greater than that of WT littermates (p<0.01,
23-29 slices/6-8 mice). In contrast, PPF in slices from young Df1(16)/+
mice was not elevated compared to that in slices from WT littermates
(p>0.05, 18-19 slices/5-6 mice). Using whole-cell recordings in mature
animals, EPSC facilitation evoked by trains of synaptic stimulations was
measured. Synaptic facilitation during high-frequency stimulation was
also substantially enhanced in mature but not young mutant mice. Thus,
synaptic facilitation was significantly increased in mature Df(16)1/+
mice compared to WT littermates if induced by a 50-Hz stimulation train
(p<0.05, 10-17 neurons) or a 100-Hz (p<0.05, 9-17 neurons)
stimulation train. In contrast, no enhancement of synaptic facilitation
was observed in younger Df(16)1/+ mice (p>0.05, 6-8 neurons). Thus,
both forms of STP were enhanced in mature mutant mice compared to WT
littermates, indicating that presynaptic function becomes enhanced in
Df1(16)/+ mice upon maturation.

[0082] To further distinguish between contributions from presynaptic and
postsynaptic loci to the LTP phenotype of mature Df1(16)/+ mice, Ca2+
transients were compared in dendritic spines of CA1 neurons in response
to the LTP-induction protocol (40 stimulations at 200 Hz) delivered by
either synaptic stimulation (tetanus) or two-photon photolysis of caged
glutamate. The comparison of the results from these two methods allows
for the distinction between presynaptic and postsynaptic function,
because two-photon glutamate uncaging (TGU) releases exogenous glutamate,
thereby bypassing the release of endogenous neurotransmitters from
presynaptic terminals (Matsuzaki, et al. (2001) Nat. Neurosci.
4:1086-1092). To perform these experiments, CA1 neurons were filled with
the Ca2+ indicator Fluo 5F and the fluorescent dye ALEXA 595 through a
whole-cell pipette and changes in Fluo 5F fluorescence in dendritic
spines in current-clamp mode in response to either synaptic stimulation
or TGU was measured. To mimic synaptically evoked mEPSPs, the uncaging
laser power was adjusted to evoke 0.4- to 0.5-mV EPSPs in response to a
single TGU pulse. Ca2+ transients evoked by the 200-Hz tetanus (40
stimulations) were substantially larger in mature Df(16)1/+ mice than in
WT mice. On average, during 200-Hz tetanus, peak ΔG/R was
0.16±0.03% in Df(16)1/+ mice but only 0.10±0.02% in WT mice
(p=0.03, 12-15 neurons). In contrast, 200-Hz TGU (40 stimulations)
produced Ca2+ transients of similar amplitudes in dendritic spines of
Df(16)1/+ and WT mice (p=0.21, 7-11 neurons). Kinetics of postsynaptic
Ca2+ transients in response to 40 synaptic or 40 TGU stimulations were
similar between Df(16)1/+ and WT mice. Rise times (10%-90%) of Fluo 5F
fluorescence changes were 134.4±6.6 ms in Df1(16)1/+ mice and
136.9±12.0 ms in WT mice (12-15 neurons) in response to synaptic
stimulation and 168.4±13.9 ms and 153.4±4.2 ms (7-11 neurons),
respectively, in response to TGU (p=0.139, one-way ANOVA). Decay times
(90%-10%) were 857.4±60.1 ms in Df(16)1/+ mice and 758.3±62.0 ms in
WT mice during synaptic stimulation (12-15 neurons) and 907.3±125.1 ms
and 895.4±104.5 ms (7-11 neurons), respectively, during TGU (p=0.790,
one-way ANOVA). Excitatory postsynaptic potentials evoked by 200 Hz TGU
(uEPSPs) in these experiments were also similar between genotypes
(p=0.765, 7-11 neurons). Similarly, in voltage-clamp experiments, 40 TGU
stimulations delivered at 200 Hz to dendritic spines evoked uEPSCs of
similar amplitudes (p=0.257, 5-6 neurons) in Df(16)1/+ and WT mice. In a
similar fashion, no differences in amplitudes or kinetics of postsynaptic
Ca2+ transients were detected between genotypes in response to a single
TGU stimulation. Because no difference in calcium transients were
detected between genotypes in TGU experiments, it was determined whether
glutamatergic receptors and the calcium indicator were saturated during
these experiments. Longer TGU stimulations (80 or 120 pulses) delivered
at 200 Hz produced significantly larger calcium transients in dendritic
spines than did 40 TGU stimulations (p<0.05, 10 neurons). This result
indicated that neither glutamatergic receptors nor the calcium indicator
were saturated during the TGU experiments. Together, these findings
indicate that the Df(16)1 microdeletion does not affect postsynaptic
neurons but enhances neurotransmitter release from presynaptic terminals
during high-frequency synaptic activity.

[0084] Because previous experiments suggested involvement of enhanced
neurotransmitter release in the LTP phenotype in mature Df(16)1/+ mice
during high-frequency activity, a more direct method was employed. For
this purpose, neurotransmitter release was compared in individual
presynaptic boutons using the FM 1-43 dye-unloading assay in acute
hippocampal slices. It has been shown that this assay can reliably
measure the rate of neurotransmitter release from presynaptic terminals
(Zakharenko, et al. (2001) supra; Zakharenko, et al. (2002) supra).
Presynaptic CA3 terminals were labeled by electrically stimulating
Schaffer collaterals at 10 Hz in the presence of the NMDA receptor
blocker D-APV (50 μM) and the fluorescent dye FM 1-43 (10 μM).
After washing out the extracellular dye, fluorescent puncta, representing
stimulated presynaptic boutons, were observed. A subsequent synaptic
stimulation mimicking the LTP-induction protocol and delivered as 10
trains of 40 stimulations at 200 Hz, unloaded the dye from these
presynaptic boutons. FM 1-43 destaining from each fluorescent punctum was
fitted with a single exponential decay, and destaining rates were
calculated. It was found that the average rate of FM 1-43 destaining,
which was measured as the reciprocal value of FM 1-43 destaining
half-times (1/t1/2) was faster for boutons from Df(16)1/+ mice
(0.0562±0.0022 s-1) than for boutons of WT mice (0.0481±0.0019 s-1,
n=7 slices for both genotypes, 17-67 boutons per slice, p=0.019). These
results confirm that evoked neurotransmitter release, rather than
postsynaptic function, is enhanced in the Df(16)1/+ mouse model of
22q11DS.

[0086] Evoked neurotransmitter release depends on the concentration of
Ca2+ inside presynaptic terminals. To test whether presynaptic Ca2+ is
responsible for the observed augmentation of neurotransmitter release,
Ca2+ transients were measured in presynaptic terminals of CA3 pyramidal
neurons from mature Df(16)1/+ and WT mice. Cells were filled with ALEXA
594 and Fluo 5F, and presynaptic boutons were identified along axons.
Axons were distinguished from dendrites by their thinner diameter and the
lack of dendritic spines. Calcium transients were detected in individual
boutons in response to an AP triggered by injecting a step of
depolarizing current through a whole-cell pipette. It was found that a
single AP triggered Ca2+ transients of similar amplitudes in presynaptic
boutons of mature Df(16)1/+ and WT mice (p=0.66, 34-36 boutons from 10-12
neurons). Rise times (10%-90%) of Ca2+ transients were also
indistinguishable between presynaptic boutons of mutant (2.35±0.18 ms,
n=34) and WT mice (2.41±0.15 ms, n=36, p=0.78). However, decays of
Ca2+ transients (90%-10%) were somewhat slower in boutons of Df(16)1/+
mice (114.26±15.07 ms, n=34) than in WT littermates (73.22±7.34 ms,
n=36, p=0.036). This modest alteration in presynaptic Ca2+ kinetics was
substantially exacerbated when Ca2+ transients were evoked with the
LTP-induction protocol (40 APs delivered at 200 Hz). Similar to the
single-AP experiment, the rise of Ca2+ transients showed no detectable
difference between the genotypes (rise times (10%-90%), 91.6±2.9 ms
and 91.5±2.3 ms, respectively, p=0.79, 40 boutons/15 neurons per
genotype). However, the decays (90%-10%) of Ca2+ transients evoked by 40
APs were significantly slower in boutons of mature Df(16)1/+ mice
(1491.2±117.5 ms, n=40) than in WT mice (886.1±91.8 ms, n=40,
p<0.001). The amplitude of Ca2+ transients evoked by the LTP-induction
protocol was also increased in Df(16)1/+ mice. Peak amplitude of Fluo 5F
fluorescence evoked by 40 APs in presynaptic terminals of CA3 neurons was
approximately 30% higher in Df(16)1/+ mice than in WT littermates
(p<0.001, n=40 per genotype). These data indicate a strong
dysregulation of Ca2+ dynamics in presynaptic terminals of Df(16)1/+
mutants.

[0087] The AP-evoked rise in Ca2+ concentration in presynaptic terminals
occurs via activation of voltage-gated Ca2+ channels and is augmented
through the release of Ca2+ from internal stores (Emptage, et al. (2001)
Neuron 29:197-208). The observations that EPSCs evoked by a single
synaptic stimulation and that rise times of presynaptic Ca2+ transients
are normal in Df(16)1/+ mice strongly argued against the notion that Ca2+
influx through voltage-gated Ca2+ channels is affected in these mutants.
Therefore, attention was focused on internal Ca2+ stores, which are
filled primarily through SERCA-mediated mechanisms. To test whether the
level of SERCA is altered in Df(16)1/+ mice, western blot analysis of
hippocampal extracts was used to compare protein levels of SERCA2, the
only SERCA isoform expressed in the forebrain (Baba-Aissa, et al. (1998)
Mol. Chem. Neuropathol. 33:199-208).

[0088] Quantification of western blots revealed that the level of SERCA2
protein was increased approximately 20% in whole-tissue lysates of mutant
hippocampus compared to that of WT littermates (p=0.006, 9 mice per
genotype). This difference was not observed in younger mice (p=0.84, 8-10
mice), indicating that a dysregulation of SERCA expression occurs with an
age dependence similar to that of the LTP and spatial memory phenotypes.
SERCA2 transcript levels, measured by quantitative real-time PCR, were
unaltered, indicating that dysregulation of SERCA2 occurs only at the
protein level. This is consistent with results from microarray studies of
22q11DS models, which have shown no alterations in SERCA2 transcript
levels (Jurata, et al. (2006) Schizophr. Res. 88:251-259; Prescott, et
al. (2005) Hum. Genet. 116:486-496; Stark, et al. (2008) supra). Because
mature Df(16)1/+ mice show an enhancement in synaptic plasticity, it was
determined whether the level of SERCA2 is increased in the synapses of
these mutants. The level of SERCA2 protein was significantly higher in
hippocampal synaptosomes of mature Df(16)1/+ mice than in synaptosomes
from WT littermates (p<0.001, 8-10 mice).

[0090] To test whether SERCA influenced the enhanced neurotransmitter
release and LTP in mature Df(16)1/+ mice, the rates of FM 1-43 destaining
were measured in the presence of CPA (50 μM), an inhibitor of SERCA
pumps that depletes internal Ca2+ stores. The addition of CPA eliminated
the difference in FM 1-43 destaining between mature Df(16)1/+ and WT
mice. In the presence of CPA, the rate of FM 1-43 destaining in Df(16)1/+
mice elicited with 200-Hz tetanus trains reached 0.0467±0.0026 s-1
(n=10 slices, 21-84 boutons per slice), which was significantly slower
than that in the absence of CPA (p=0.02, 7 slices). In contrast, in WT
mice the rates of FM 1-43 destaining were similar in the presence
(0.0479±0.0027 s-1, n=8 slices, 13-46 boutons per slice) or absence
(n=7, p=0.96) of CPA. Interestingly, in the presence of CPA, the rates of
FM 1-43 destaining from presynaptic boutons in Df(16)1/+ and WT mice did
not differ (p=0.76); thus, CPA rescued enhanced neurotransmitter release
in Df(16)1/+ mice. No significant difference in decay (p=0.22) or
amplitude (p=0.72) of presynaptic Ca2+ transients evoked by 40 APs (200
Hz) was also found in the presence of CPA in WT and Df(16)1/+ mice (14-19
boutons, 5 neurons per genotype). These data indicate that blocking SERCA
with CPA rescued presynaptic calcium dysregulation in mutant mice. In a
similar fashion, CPA rescued the increase in PPF in Df(16)1/+ mice. Thus,
PPF measured at 20- and 50-ms intervals in Df(16)1/+ mice was
significantly reduced from 2.09±0.15 and 2.01±0.09, respectively,
in the absence of CPA to 1.78±0.13 (p=0.001, 28 slices) and
1.76±0.09 (p=0.019, 28 slices) when CPA was added to the bath
solution. In contrast, in WT mice PPF was similar in the presence
(1.64±0.11 for 20-ms and 1.61±0.05 for 50-ms intervals, 19 slices)
and in the absence of CPA (1.58±0.08 for 20-ms, p=0.49 and
1.69±0.05 for 50-ms intervals, p=0.62, 19 slices). Importantly, no
significant difference in PPF was found between the genotypes in the
presence of CPA (p=0.283 for 20-ms interval and p=0.455 for the 50-ms
interval, 19-28 slices). Together, these data indicate that the depletion
of internal Ca2+ stores rescued the enhancement of presynaptic Ca2+
transients and neurotransmitter release in mature Df(16)1/+ mice. They
also predict that the depletion of Ca2+ stores by SERCA inhibitors should
rescue the LTP enhancement in mature Df(16)1/+ mice.

[0091] To test this, LTP induced by 200-Hz tetanization was measured in
the presence of either CPA or thapsigargin (4 μm), another SERCA
inhibitor. Both agents rescued the enhancement of PTP and LTP in
Df(16)1/+ mice. In slices from CPA-treated Df(16)1/+ mice, fEPSP5 was
reduced to 45% (p=0.0001), and fEPSP360 was reduced to 60% of that
detected in vehicle-treated Df(16)1/+ mice (p=0.021, 16-23 slices, 7-8
mice). Similarly, thapsigargin reduced the fEPSP5 and fEPSP360 in slices
from Df(16)1/+ mice to 69% (p=0.015) and 57% (p=0.005), respectively, of
that in vehicle-treated Df(16)1/+ slices (22-23 slices, 8-10 mice).
Interestingly, neither SERCA inhibitor affected WT LTP induced by the
200-Hz induction protocol. This is consistent with results showing that
SERCA inhibitors affect LTP induced only by weak (but not strong, e.g.,
200 Hz) stimulation protocols (Behnisch & Reymann KG (1995) Neurosci.
Lett. 192:185-188; Matias, et al. (2002) Neuroreport 13:2577-2580; Zhang,
et al. (2009) Nature 460:632-636). Thus, in the presence of CPA, WT
fEPSP5 (p=0.210) and fEPSP360 (p=0.680) were not significantly different
from those seen in the absence of CPA (16-52 slices, 6-15 mice).
Similarly, no significant difference was seen between fEPSP5 (p=0.384)
and fEPSP360 (p=0.939) in the presence or absence of thapsigargin (22-52
slices, 10-mice). Importantly, in the presence of CPA and thapsigargin,
the increases in fEPSPs at either time point were not significantly
different between mature Df(16)1/+ and WT mice (p=0.951 and p=0.436 for
fEPSP5, and p=0.297 and p=0.623 for fEPSP360, respectively), indicating
that inhibition of SERCA rescued the LTP phenotype in Df(16)1/+ mice.

The results herein demonstrate that within 16 to 20 weeks of birth the
Df(16)1/+ mouse model of 22q11DS develops a substantial enhancement in
LTP that coincides with a deficit in spatial memory. Furthermore, the
increase in LTP caused by the hemizygous deletion of 22q11DS-related
genes is due to enhanced glutamate release from presynaptic terminals and
not due to changes in postsynaptic structures or function. Moreover, it
was demonstrated that SERCA2 upregulation is a contributor to the
dysregulation of presynaptic Ca2+, enhanced glutamate release, and
increased LTP in the model of 22q11DS.